TABLE OF CONTENTS . . .
1. FIELD OF THE INVENTION . . .
1.1 BRIEF DESCRIPTION OF THE INVENTION . . .
1.2 BACKGROUND OF THE INVENTION . . .
2. SUMMARY OF THE INVENTION . . .
3. BRIEF DESCRIPTION OF THE DRAWINGS . . .
4. DETAILED DESCRIPTION OF THE INVENTION . . .
5. EXAMPLES . . .
6. REFERENCES . . .
THE CLAIMS . . .
ABSTRACT . . .
1.1 Brief Description of the Invention
The invention disclosed herein relates to a new gene probe biosensor employing near field surface enhanced Raman scattering (NFSERS) for direct spectroscopic detection of DNA hybridization without the need for labels, and the invention also relates to methods for using the biosensor.
1.2 Background of the Invention
In 1928, C. V. Raman and his collaborator, K. S. Krishnan, established that the spectrum of inelastically scattered light can provide a unique fingerprint of molecular structure. Since this initial discovery, Raman spectroscopy has advanced dramatically. Many Raman-related analytical instruments have been developed, some of which have applicability to proteins and nucleic acids. Recent developments have enabled the use of Raman spectroscopy to obtain information such as conformation and/or orientation of molecules and some molecular groups, local hydrogen bonding interactions, and time dependence of structural or organizational properties. Thomas, G. J., xe2x80x9cRaman Spectroscopy of Protein and Nucleic Acid Assemblies,xe2x80x9d Annu. Rev. Biophys. Biomol. Struct. 28:1-27 (1999).
The discrete vibrational energies (Raman band frequencies), scattering probabilities (Raman intensities) and tensor characteristics (Raman polarizations) that constitute the Raman spectra are a function of molecular geometry and intra- and intermolecular force fields.
Early experimental work in the field of Raman spectroscopy demonstrated the advantages of surface-enhanced Raman scattering (SERS) as a technique for detecting and identifying molecules. See Cotton, T. M. xe2x80x9cApplication of Surface-Enhanced Raman Spectroscopy to Biological Systemsxe2x80x9d J. Raman Spect. 23: 729-742 (1991). For example, between 1974 and 1977, several researchers showed that Raman scattering from pyridine on a roughened silver electrode was enhanced by approximately six orders of magnitude. Id. SERS has been used to study various types of amino acids and peptides on silver surfaces, as well as to study the behavior of DNA at silver colloids.
Surface enhanced Raman scattering has also been investigated as a method for detecting and identifying single base differences in double stranded DNA fragments. Chumanov, G. xe2x80x9cSurface Enhanced Raman Scattering for Discovering and Scoring Single Based Differences in DNAxe2x80x9d Proc. Volume SPIE, 3608 (1999).
SERS has also been used for single molecule detection. Kneipp, K. xe2x80x9cSingle Molecule Detection Using Surface-Enhanced Raman Scattering (SERS)xe2x80x9d Physical Review Letters 78(9):1667-1670 (1997). SERS results in strongly increased Raman signals from molecules which have been attached to nanometer sized metallic structures.
SERS principles have also been used in the development of gene probes which do not require the use of radioactive labels. These probes can be used to detect DNA via hybridization to a DNA sequence complementary to the probe. Vo-Dinh, T. xe2x80x9cSurface-Enhanced Raman Gene Probesxe2x80x9d Anal. Chem. 66:3379-3383 (1994).
The Human Genome Project and other recent advances in molecular biology have spurred the development of new methods for the labeling and detection of DNA and DNA fragments.
Traditionally, radioisotopes have been used as labels for DNA. More recently, fluorescent, chemiluminescent and bioactive reporter groups have been used. The reporter groups are typically incorporated in the primers or the deoxynucleoside triphosphates to label the newly synthesized DNA fragments. The DNA fragments of interest are allowed to hybridize to a set of bound or immobilized DNA fragments.
Among the various methods for identifying genes, the most widely used are technologies which require radioactive labels. A variety of disadvantages are associated with the use of radioactive labels, including the short shelf life of common labels and the safety hazards associated with the use of radioactive compounds. Accordingly, there is a strong need in the art for a method for identifying genes which does not require the use of radioactive labels.
Methods for manufacturing oligonucleotide, DNA and protein microchips and microarrays are known in the art. Research is ongoing into the use of such microchips and microarrays in DNA and RNA sequence analysis, diagnostics of genetic disease, gene polymorphisms studies, and analysis of gene expression. Microchips have been developed in which oligonucleotides are immobilized within polyacrylamide gel pads. Robotics can be employed for the manufacture of microchips containing thousands of immobilized compounds.
Various attempts have been made to enable the sequencing of DNA without the necessity of using radioisotopes, or fluorescent substances. For example, U.S. Pat. No. 5,821,060 describes a process for DNA sequencing, mapping and diagnostics which utilizes the differences between the chemical composition of DNA and that of peptide nucleic acid sequences (PNAs) to provide DNA sequencing, mapping or diagnostics using natural DNA fragments. The process includes the steps of hybridizing PNA segments to complementary DNA segments which are affixed to a hybridization surface, or hybridizing in DNA segments to complementary PNA segments which are fixed to a hybridization surface and using mass spectrometric or non-mass spectrometric techniques to analyze the extent of hybridization at each potential hybridization site.
It is a an object of the present invention to provide molecular sequencing, mapping, screening, diagnostic process and other molecular hybridization processes, in which normal, unlabled DNA is used rather than DNA labeled with stable isotopes, radioactive isotopes or fluorescent groups, and which provides superior spectral specificity as compared to methods of the prior art. Achieving this object will eliminate some of the expensive reagents and labor involved in the labeling of DNA and thereby significantly reduce time, effort and expense of DNA analysis, while enabling highly accurate DNA sequencing, mapping, screening, diagnostic and other molecular hybridization related processes.
In some cases, polymorphisms comprise mutations that are the determinantive characteristic in a genetic disease (hemophilia, sickle-cell anemia, etc.). A xe2x80x9cpolymorphismxe2x80x9d is a variation in the DNA sequence of some members of a species. A polymorphism is said to be xe2x80x9callelicxe2x80x9d because some members of a species have the mutated sequence, while other members have the non-mutated sequence. Single nucleotide polymorphisms (SNPs) contain a polymorphic site. A variety of methods have been developed for the characterization of SNPs. Such methods include, for example, the direct or indirect sequencing of the site, the use of restriction enzymes with specificity for the allelic site to create or destroy a restriction site, the use of allele-specific hybridization probes, the use of antibodies that are specific for the proteins encoded by the different alleles of the polymorphism, and other biochemical techniques. It is an object of the present invention to provide advanced surface detection methods which enable the characterization of SNPs without the necessity for the use of restriction enzymes which affect the SNP site, without the necessity for allele-specific hybridization probes, and without the necessity of using antibodies specific for the proteins encoded by the different alleles of the polymorphism.
Other objects and advantages of the present invention over the prior art will become apparent to those skilled in the art upon review of the detailed description that follows.
The applicant has surprisingly and unexpectedly discovered, using a novel analytic technique, coupling near-field optics with SERS techniques, that each hybridization member in a hybridized pair of molecules (e.g., hybridized DNA fragments) has a unique spectrum of low frequency (lattice-type) vibrations. The novel analytic technique presented herein is employed in the novel spectroscopic instrument of the present invention, which is useful for detecting molecular hybridization. The novel instrument and methods presented herein enable vastly improved spectral sensitivity as compared to known methods.
One object of the present invention is to provide a more efficient, reliable, faster and more accurate method for direct detection of nucleic acid hybridization on high density nucleic acid chips. The invention provides direct spectroscopic detection of DNAxe2x80x94DNA, DNAxe2x80x94RNA, and RNAxe2x80x94RNA hybridization.
Among the many advantages of the apparatus and method of the present invention, are the ability to eliminate the need for labeling (by fluorescent or other labels) as is required in currently used methods. Furthermore, the apparatus and method of the present invention enable high throughput screening of DNA without the necessity for PCR amplification.
The invention provides an analytical method for determining whether a DNA sample comprises double-stranded DNA, said method comprising analyzing the DNA sample by near field Raman spectroscopy to determine whether the sample produces lattice vibrations, wherein the presence of lattice vibrations indicates the presence of double stranded DNA in the DNA sample. In a preferred aspect, the DNA sample is associated with a substrate, e.g., a substrate selected from the group consisting of: nucleic acid chips, peptide nucleic acid chips, conducting carbon nanotube plates; microfluidic nucleic acid chips.
The invention also provides a spectroscopic system for detecting molecular hybridization, said system comprising a near-field SERS substrate arranged to support one or more predetermined hybridizeable molecules thereon; a coherent radiation source arranged to impinge coherent radiation onto each of the hybridizeable molecules to responsively produce a pattern of scattered photons; a photonic collector arranged in photon-gathering relationship to the scattered photons and adapted to transmit the gathered scattered photons; a Raman spectrograph arranged in photon receiving relationship to the photonic collector and adapted to generate an output correlative to the collected scattered photons transmitted by the photonic collector; and a spectral to electronic converter, arranged to receive the output of the Raman spectrograph and to convert same to an electronic output indicative of the presence or absence of hybridized molecules on the SERS substrate.
In another embodiment, the near field SERS substrate is selected from the group consisting of: nucleic acid chips, peptide nucleic acid chips, conducting carbon nanotube plates, microfluidic nucleic acid chips, optical nanocluster microchips, plates coated with colloid silver, plates coated with colloid gold, plates coated with colloid platinum, and conducting carbon nanotube plates. The near field SERS substrate is a preferably a microchip or microarray.
The one or more predetermined hybridizeable molecules disposed on the near field SERS substrate are preferably ssDNA or ssRNA.
The laser light source is preferably selected from the group consisting of: argon ion lasers, infrared lasers, and ultraviolet lasers.
The spectral to electronic converter preferably comprises a CCD array and the photonic collector optionally comprises an ICCD array.
The present invention also provides a method for detecting hybridized DNA comprising providing the spectroscopic system described above; exposing the predetermined hybridizeable molecules disposed on the near field SERS substrate to a sample containing one or more sample molecules having the capacity to hybridize to the predetermined hybridizeable molecules; directing the laser beam from the laser light source onto each of the one or more predetermined hybridizeable molecules to create a pattern of scattered photons for each of said hybridizeable molecules; collecting the scattered photons for each of said hybridizeable molecules and directing them to a Raman spectrograph; collecting photonic data from the Raman spectrograph and transforming said photonic data into electronic data for further data processing; and determining whether each of the hybridizeable molecule is hybridized to a sample molecule by comparing the Raman spectrum of (i) each hybridizeable molecule exposed to the sample to (ii) the Raman spectrum to the corresponding unhybridized predetermined hybridizeable molecule.
Other aspects of the invention will become apparent to those of skill in the art from the drawings of FIGS. 1-4 and the Brief Description of the Drawings presented in Section 3 hereof, from the Detailed Description of the Invention in Section 4 hereof, and from the Examples, presented in Section 5 hereof.